SUMMARY/ABSTRACT The new clinical guidelines for diagnosing Fetal Alcohol Spectrum Disorders (FASD) list self-regulation as one of the key behavioral deficits in children affected by prenatal alcohol exposure (PAE). There is a fundamental gap in knowledge about the underlying mechanisms, spectrum, and severity of such deficits early in life and the best analytical approaches to identify them. In addition, the effect of prenatal stress and postnatal environment on PAE-induced alterations is poorly understood. In this renewal application of the Ethanol, Neurodevelopment, Infant, and Child Health (ENRICH) study, we seek continuous support for our established recruitment/retention pipeline, and propose new highly innovative studies of stress reactivity/regulation in infants with PAE. The long-term goal is to identify indices of atypical brain development following PAE as early as possible to enable early interventions. The objective of this application is to continue our focus on moderate PAE and to evaluate PAE effects on infant stress reactivity/regulation and the mechanisms underpinning altered hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS) functioning. We will evaluate the relationship between PAE (exposure of interest), prenatal stress (moderator), biological measures of HPA axis (mediators), and physiological and behavioral measures of stress reactivity/regulation in infants (outcomes). The rationale for this proposal is driven by the conviction that HPA and ANS dysregulation leading to altered stress reactivity/regulation in children with PAE might be the basis for some of the key PAE-induced behavioral deficits, and increased vulnerability to secondary disabilities. The central hypothesis is that PAE will be associated with heightened infant stress reactivity and poorer self-regulation (beyond the effect of prenatal stress) through fetal programming of the HPA axis. This hypothesis has been formulated on the basis of preclinical data at UNM and clinical data from the current funding cycle of ENRICH-1 and will be tested by pursuing three specific aims, which evaluate the contributing effects of PAE on 1) Programming of the fetal HPA axis, assessed as expression of key placental and umbilical cord markers of HPA axis; 2) Infant physiological reactivity (heart rate variability [HRV]) dynamic changes during basal-stressor-recovery periods assessed in the newborn period and at 6-months of age; 3) Infant behavioral reactivity and regulation assessed in the newborn period and at 6-months of age. The comprehensive multi-systemic approach employed in this study is highly innovative and has not previously been used. Innovation is further driven by our focus on moderate PAE and ability to assess the trajectory of impaired stress reactivity/regulation (newborn, 6-months evaluations) in a large prospective cohort study. This research is significant because it involves comprehensive repeated-measures assessment of neuroendocrine, electrophysiological, and behavioral indices of impaired stress reactivity/regulation, which will lead to refinement of analytical techniques for accurate identification of PAE deficits in infancy before higher-order behavioral deficits manifest.